Type 4 piliated bacteria are involved in a variety of ophthalmic and other disease. Bacteria known to express type 4 pili include Moraxella bovis, Neisseria gonorrhoeae, Bacteroides nodosus, Pseudomonas aeruginosa and Vibrio cholerae. Type 4 pili are related both in terms of amino-terminal amino acid sequence homology and antibody cross-reactivities, and also functionally since cloning just the pilin genes of B. NOdosus or M. bovis into P. aeruginosa allow expression of B. nodosus or M. bovis pili. The goal of this project is to understand the genetics and molecular biology of type 4 pili, particularly those on bacteria of the genera Moraxella and Kingella. M. bovis and M. lacunata pilin gene expression is regulated by the inversion of a 2 kb region of DNA, which in M. bovis Epp63 results in the expression of either Q or I pili. We plan to further investigate the mechanisms of this pilin gene inversion event, and ultimately to carry out the inversion event in an in vitro system of completely define components. Sequence analysis of polymerase chain reaction (PCR) amplified DNA from nonpiliated (P-) variants will be used to determine the mechanisms of P+ to P- switch for M. bovis and M. lacunata. We have identified an open reading frame (ORF1) whose role is as yet unknown, but it seems to be coexpressed with Q (but not I) pilin. We plan transcriptional studies to localize the pilin gene promoter, to determine whether ORF1 is coexpressed with Q-pilin, and then to isolate the protein coded for by ORF1 and determine if it is a minor structural component of the pilus, or if it has other roles in pilus biosynthesis. Genomic Southern hybridization analysis of Kingella denitrificans reveals multiple DNA fragments which hybridize to the Q-pilin gene probe. This appears similar to the hybridization patterns seen in the multi-pilin gene system of N. gonorrhoeae, and we plan to clone, analyze and map the pilin gene loci of K. denitrificans to determine if they are organized and undergo antigenic variation similar to that described for N. gonorrhoeae.